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Chemokine molecules play important roles in the immune system. However, several chemokine molecules are expressed during early development before the immune system is established. Using reverse transcription–polymerase chain reaction (RT-PCR) and overexpression of chemokine molecules, we identified and characterized Xenopus laevis CXC-type chemokine ligands (XCXCL13L1, XCXCL13L2, XCXCLa, XCXCLb, XCXCLd, and XCXCLe) and receptors (XCXCR1/2, XCXCR3, XCXCR5, XCXCR6, and XCXCRa) during early development. The CXC-type ligands have low identity with genes for human CXC ligands (CXCL). With the exception of XCXCRa, the CXC receptors (CXCR) identified in the present study had high (40%–65%) identity with human CXCR genes. Although the expression patterns for the CXCL and CXCR genes differed, transcript levels for all genes were very low during early embryogenesis. Overexpression of XCXCL13L1, XCXCL13L2, XCXCLa, XCXCR3, XCXCR6, and XCXCRa interfered with gastrulation and neural fold closure. The results of the present study suggest that several chemokine molecules are related to cell movements during early morphogenesis.
Fig. 2 (left). Expression patterns of Xenopus chemokine ligand transcripts. Reverse transcriptionolymerase chain reaction analysis was performed using total RNA extracted from Xenopus embryos at different stages of development and from different regions. Ornithine decarboxylase (ODC) was used as an internal control. (A) Temporal expression patterns. U, unfertilized eggs. The numbers indicate developmental stages. The number of polymerase chain reaction (PCR) cycles is given on the right. (B) Spatial expression patterns. Embryos were dissected at the stages indicated, and dissections were performed as shown in the bottom panels. Abbreviations: D, dorsal; Vn, ventral; A, animal; M, marginal; Vg, vegetal; H, head.
Fig. 3 (right). Expression patterns of Xenopus chemokine receptor transcripts. Reverse transcriptionolymerase chain reaction analysis was performed using total RNA extracted from Xenopus embryos at different stages of development and from different regions. Ornithine decarboxylase (ODC) was used as an internal control. (A) Temporal expression patterns. U, unfertilized eggs. The numbers indicate developmental stages. The number of polymerase chain reaction (PCR) cycles is given on the right. (B) Spatial expression patterns. Embryos were dissected at the stages indicated, and dissections were performed as shown in the bottom panels. Abbreviations as in Fig. 2.
Fig. 4. Phenotypes of Xenopus embryos injected with chemokine ligands into the dorsal side. (A) Phenotypes of the injected embryos.The mRNA of each chemokine ligand (500 pg/blastomere) was injected into two dorsal blastomeres of 4-cell-stage embryos. Left panels, Stage 12, vegetal view; center panels, Stage 16, dorsal view; right panels, Stage 30, lateral view. (B)The ratio of the injected embryos exhibiting gastrulation defects. (C) RT-PCR analysis of mesodermal marker genes. Total RNA for RT-PCR was extracted the dorsal sectors of the injected embryos at stage 10.
Fig. 5. Phenotypes of Xenopus embryos injected with chemokine receptors into the dorsal side. (A) Phenotypes of the injected embryos. The mRNA of each chemokine receptor (500 pg/blastomere) was injected into two dorsal blastomeres of 4-cell-stage embryos. Left panels, Stage 12, vegetal view; center panels, Stage 16, dorsal view; right panels, Stage 30, lateral view. (B) The ratio of the injected embryos exhibiting gastrulation defects. (C) RT-PCR analysis of mesodermal marker genes. Total RNA for RT-PCR was extracted the dorsal sectors of the injected embryos at stage 10.
Fig. 6. Phenotypes of Xenopus embryos injected with chemokine re- ceptors into the dorsal side. (A-G) Phenotypes of the injected embryos. (A) Control. (B) XCXCL13L1. (C) XCXCL13L2. (D) XCXCLa. (E) XCXCR3. (F) XCXCR6. (G) XCXCRa. (H) The ratio of the injected embryos exhibiting ventral morphogenesis defects.
